It has been shown recently that voltage-gated neuronal Na channels are modulated by heterotrimeric GTP-binding proteins. The aim of this project is to investigate the molecular mechanisms which underlie the interaction of Na channels with G proteins. The initial studies will identify the specific G protein subunit(s) responsible for channel modulation by overexpression of exogenous G protein subunits along with Na channel alpha subunits into tsA201 cells, a subclone of the human embryonic kidney cell line HEK 293. The functional consequences brought by these subunits will be examined by patch clamp recordings. Then the abilities of fusion proteins containing each of the large intracellular portions of the Na channel alpha subunits to interact with the specific G protein subunit identified above will be tested first in biochemical experiments followed by functional analysis in tsA201 cells. Once a specific interaction region has been identified, the critical amino acid residues involved in G protein binding and modulation of Na channels will be further defined by site-directed mutagenesis, first in Na channel fusion proteins and then in the Na channel itself. Determination of G protein sites on Na channels is vital to understand the molecular mechanism through which G proteins modify Na channel gating. Modulation of Na channels by G protein-coupled receptors is likely to be a potent pathway for neuronal modulation. The same receptors are likely to control several ion channels including Na channels. My results will provide new information concerning how overall neuronal activity is controlled.